CN113730398A

CN113730398A - Artesunate and new application of composition containing same

Info

Publication number: CN113730398A
Application number: CN202111165973.7A
Authority: CN
Inventors: 禹志领; 符秀琼; 金艳霞
Original assignee: Hong Kong Baptist University HKBU
Current assignee: Hong Kong Baptist University HKBU
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-03

Abstract

本发明涉及一种青蒿琥酯在制备防治耐受BRAF(V600E)抑制剂的肿瘤的效果的药物中的应用，以及人参皂苷Rg3和青蒿琥酯组合物在制备防治耐受BRAF(V600E)抑制剂的肿瘤的药物中的应用，如此能够增强对耐受BRAF(V600E)抑制剂的肿瘤的治疗效果。The invention relates to the application of artesunate in the preparation of a medicine for preventing and treating tumors resistant to BRAF (V600E) inhibitors, and the preparation of a ginsenoside Rg3 and artesunate composition for preventing and treating resistant BRAF (V600E) The application of the inhibitor in the medicine of the tumor can enhance the therapeutic effect of the tumor resistant to the BRAF (V600E) inhibitor.

Description

Artesunate and new application of composition containing same

Technical Field

The invention relates to a new application of a medicament, in particular to a new application of artesunate and a composition containing artesunate.

Background

BRAF belongs to a serine/threonine specific protein kinase and is a member of the growth signal transduction protein kinase family. The T1799A point mutation of BRAF gene (V600E mutation) can lead to the continuous activation of BRAF protein, thereby activating downstream mitogen-activated protein kinase (MAPK)/extracellular regulatory protein kinase (ERKs) signal cascade, stimulating cell proliferation and differentiation, inhibiting apoptosis and finally promoting cancer progression. This mutation occurs in a variety of cancers, such as hairy cell leukemia (about 100%), Erdheim-Chester leukemia (about 54%), papillary thyroid cancer (about 50%), melanoma (40% -60%), colorectal cancer (about 12%), and non-small cell lung cancer (about 7%), among others. Vemurafenib (Vemurafenib) is the first BRAF (V600E) inhibitor approved by the Food and Drug Administration (FDA) and is used to treat unresectable or metastatic BRAF (V600E) mutation-positive melanoma and Erdheim-Chester leukemia, but patients often develop drug resistance after receiving treatment for 5-8 months. Another BRAF (V600E) inhibitor, Dabrafenib (Dabrafenib), and also Trametinib (Trametinib) and cobitinib (Cobimetinib), inhibitors of BRAF downstream MEK, have also been FDA approved for use in BRAF (V600E) mutant melanoma, but patients are also resistant to them. The FDA subsequently approved the combination of a BRAF (V600E) inhibitor and a MEK inhibitor, including the combination trametinib/dabrafenib and the combination cobitinib/vemurafenib, for the treatment of melanoma. The curative effect is remarkable, and the median survival time of the patient reaches 22.3 months. However, the combination only delays the occurrence of drug resistance, and patients also develop drug resistance to the combination after receiving treatment for months. In addition, patients with colorectal cancer who are mutation positive for BRAF (V600E) as well as some patients with melanoma and thyroid cancer who are mutation positive for BRAF (V600E) are inherently resistant to BRAF (V600E) inhibitors. Drug resistance is a big problem in BRAF (V600E) mutation positive tumor treatment, and safe and effective drug treatment for tumor patients resistant to BRAF (V600E) inhibitors is urgently needed.

Ginsenoside Rg3(Ginsenoside Rg3) is a component of the national class I anti-tumor Chinese medicine 'Shenyi Capsule', and the molecular formula is C₄₂H₇₂O₁₃And the molecular weight is 784.30. Ginsenoside Rg3 has two optical isomers, 20-R-Rg3 (formula (I)) and 20-S-Rg3 (formula (II)). The main component of the Shenyi capsule is 20-R-Rg3, which is mainly used together with chemotherapeutic drugs clinically to improve the curative effect of the chemotherapeutic drugs on primary lung cancer and liver cancer, improve the qi deficiency symptom of tumor patients, improve the immunity of organisms, and inhibit the proliferation of tumor vascular endothelial cells and the formation of new vessels. Researches find that the ginsenoside Rg3 has good anticancer effect on colon cancer, lung cancer, liver cancer, ovarian cancer, breast cancer, melanoma, nasopharyngeal carcinoma, prostatic cancer, leukemia and the like. In addition, ginsenoside Rg3 can enhance human non-small cell lung cancer and Icotinib (Ic)otinib), reducing the drug resistance of lung cancer cells to cisplatin, and reversing the drug resistance of glioblastoma to temozolomide. This suggests that ginsenoside Rg3 has potential to overcome tumor resistance. Previous researches find that the ginsenoside Rg3 can really inhibit the proliferation of drug-resistant tumor cells of BRAF (V600E) inhibitors.

Artesunate (Artesunate) is a semisynthetic derivative of artemisinin with the molecular formula C₁₉H₂₈O₈The molecular weight is 384.42, and the structural formula is shown in formula (III). The artesunate mainly acts on anaplasma viviparous of plasmodium, kills plasmodium in erythrocyte, and has antimalarial effect. The artesunate is suitable for clinically rescuing cerebral malaria and various critical malaria, and has small toxic and side effects. Researches find that the artesunate has cytotoxicity effect on B cell lymphoma, ovarian cancer, breast cancer, colorectal cancer, melanoma, nasopharyngeal carcinoma and other tumor cells, and has low toxicity on normal cells. At present, the effect of artesunate on the BRAF (V600E) inhibitor drug-resistant tumor or the effect of the ginsenoside Rg3 and artesunate composition on the BRAF (V600E) inhibitor drug-resistant tumor is not reported.

Disclosure of Invention

Based on the above, the application of the artesunate in preparing the medicine for preventing and treating the tumor resistant to the BRAF (V600E) inhibitor and the application of the ginsenoside Rg3 and artesunate composition in preparing the medicine for preventing and treating the tumor resistant to the BRAF (V600E) inhibitor can enhance the treatment effect on the tumor resistant to the BRAF (V600E) inhibitor.

In the first aspect of the invention, the application of artesunate or derivatives thereof in preparing a medicament for preventing and treating tumors resistant to BRAF (V600E) inhibitors is provided.

In one embodiment, the derivative of artesunate is TF27 (trimeric derivative of artesunate).

In a second aspect of the invention, the application of the composition consisting of the ginsenoside Rg3 or the derivative thereof and the artesunate or the derivative thereof in preparing a medicament for treating the effect of treating tumors resistant to the BRAF (V600E) inhibitor is provided.

In one embodiment, the ginsenoside Rg3 is 20-S-Rg3 and/or 20-R-Rg 3.

In one embodiment, the derivative of ginsenoside Rg3 is 20(R) -25-OH-ginsenoside Rg3 or dihydroginsenoside Rg 3.

In one embodiment, the composition comprises, in mass percent: 0.1 to 99.9 percent of ginsenoside Rg3 and/or derivatives thereof, and 99.9 to 0.1 percent of artesunate and/or derivatives thereof.

In one embodiment, the medicament further comprises pharmaceutically acceptable excipients.

In one embodiment, the dosage form of the medicament is tablets, capsules, granules, suspensions, oral liquids, injections or ointments.

In one embodiment, the medicament further comprises an anti-cancer drug.

In one embodiment, the tumor that is resistant to a BRAF (V600E) inhibitor is melanoma, large bowel cancer, prostate cancer, thyroid cancer, non-small cell lung cancer, serous ovarian cancer, or hairy cell leukemia that is intrinsically or acquired resistant to an inhibitor targeting BRAF (V600E) or to an inhibitor targeting MEK downstream of BRAF.

The invention provides two medicines for preparing a medicine for preventing and treating tumors resistant to a BRAF (V600E) inhibitor, which comprise artesunate and a composition of ginsenoside Rg3 and artesunate. Experiments prove that in a cell model, the artesunate can inhibit the drug-resistant melanoma cell proliferation of the BRAF (V600E) inhibitor by singly using the artesunate and combining the artesunate with the ginsenoside Rg3, the ginsenoside Rg3 and the artesunate have synergistic effect, and the drug treatment has no obvious toxicity to normal cells; in a mouse model, the composition of the ginsenoside Rg3 and the artesunate can obviously inhibit the growth of melanoma which is resistant to a BRAF (V600E) inhibitor, and does not affect the weight of a mouse. Artesunate and the composition of the artesunate and the ginsenoside Rg3 can be used for preparing medicines for preventing and treating tumors which are resistant to BRAF (V600E) inhibitors.

Drawings

FIG. 1 shows the effect of ginsenoside Rg3 and artesunate alone or in combination on the proliferation of melanoma cells resistant to BRAF (V600E) inhibitors; p <0.05, P <0.01, compared to the solvent control; delta represents the synergistic effect of the drug combination, and the CDI is less than 1; delta indicates that the drug combination has obvious synergistic effect, and the CDI is less than 0.7;

FIG. 2 shows the effect of the combination of ginsenoside Rg3 and artesunate on the survival rate of normal cells;

fig. 3 is a graph of the effect of ginsenoside Rg3 and artesunate alone and in combination on melanoma growth with a lotus tolerant BRAF (V600E) inhibitor in a mouse model: (A) mouse tumor pictures; (B) mouse tumor weight; (B) mouse tumor volume growth curve; p <0.05 compared to control.

Detailed Description

The novel use of the artesunate and the composition comprising the same according to the present invention will be described in further detail with reference to the following examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein, the term "and/or", "and/or" includes any one of two or more of the associated listed items, as well as any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all combinations of the associated listed items.

In the present invention, "first aspect", "second aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor are they to be construed as implicitly indicating the importance or quantity of the technical features indicated. Also, "first," "second," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed to constitute a closed limitation to the number.

In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.

In the present invention, the numerical intervals are regarded as continuous, and include the minimum and maximum values of the range and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.

The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.

The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.

"pharmaceutically acceptable" in the context of the present invention refers to those ligands, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for administration to a patient and commensurate with a reasonable benefit/risk ratio.

The invention provides an application of artesunate or derivatives thereof in preparing a medicament for treating tumors resistant to BRAF (V600E) inhibitors.

In some specific examples, the derivative of artesunate is TF27 (trimeric derivative of artesunate) or other derivatives.

The invention also provides application of the composition consisting of the ginsenoside Rg3 or the derivative thereof and the artesunate or the derivative thereof in preparing a medicine with the effect of treating tumors resistant to BRAF (V600E) inhibitors.

In some specific examples, the ginsenoside Rg3 is 20-S-Rg3 and/or 20-R-Rg 3.

In some specific examples, the derivative of ginsenoside Rg3 is 20(R) -25-OH-ginsenoside Rg3, dihydrogensenoside Rg3, or other derivatives.

In some specific examples, the composition comprises, in mass percent: 0.1 to 99.9 percent of ginsenoside Rg3 and/or derivatives thereof, and 99.9 to 0.1 percent of artesunate and/or derivatives thereof. Further, the composition comprises the following components in percentage by mass: 55 to 65 percent of ginsenoside Rg3 and/or derivatives thereof, and 45 to 35 percent of artesunate and/or derivatives thereof. Still further, the composition comprises, in mass percent: 60 to 65 percent of ginsenoside Rg3 and/or derivatives thereof, and 40 to 35 percent of artesunate and/or derivatives thereof. In some specific examples, the composition comprises, in mass percent: 25 to 30 percent of ginsenoside Rg3 and/or derivatives thereof, and 75 to 70 percent of artesunate and/or derivatives thereof.

In some specific examples, the medicament further comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include, but are not limited to, carriers, adjuvants, and the like. Wherein "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted beta-cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered gum tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

In some specific examples, the medicament can be prepared into any one dosage form of tablets, capsules, granules, suspensions, oral liquids, injections, pastes and the like. The above-mentioned drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field, and the present invention is not limited by the above-mentioned dosage forms.

In some specific examples, the drug may be administered orally, by injection, or topically, and the route of administration is not limited by the above examples.

In some specific examples thereof, the drug further comprises an anti-cancer drug. The anticancer drug is selected from one or more of Vemurafenib (Vemurafenib), Dabrafenib (Dabrafinib), Trametinib (Trametinib), cobitinib (Cobimetinib) and other BRAF (V600E) inhibitors and inhibitors targeting downstream MEK of BRAF.

In some specific examples, the tumor that is resistant to a BRAF (V600E) inhibitor is melanoma, large bowel cancer, prostate cancer, thyroid cancer, non-small cell lung cancer, serous ovarian cancer, or hairy cell leukemia that is mutation positive for an inhibitor targeting BRAF (V600E) or an inhibitor targeting MEK downstream of BRAF that is intrinsically or acquired resistant.

Specific examples are as follows.

The ginsenoside 20-R-Rg3 used in the examples was purchased from Nanjing Biotechnology Ltd (China);

artesunate is purchased from Dowmatte Biotech limited (China) and has a purity of more than 98% as determined by HPLC.

Example 1 Effect of ginsenoside Rg3 and Artesunate on proliferation of melanoma cells resistant to BRAF (V600E) Targeted drugs alone and in combination

1.1 Experimental materials

(1) Ginsenoside 20-R-Rg 3: purchased from Nanjing Biotech Ltd; weighing a certain amount of ginsenoside 20-R-Rg3, dissolving in dimethyl sulfoxide (DMSO) to obtain 20mM Rg3 solution (mother liquor), packaging, and freezing at-20 deg.C.

(2) Artesunate: purchased from Kyormant Biotech, Inc.; weighing a certain amount of artesunate, dissolving with DMSO to obtain 50mM artesunate solution (mother liquor), subpackaging, and placing in a refrigerator at-20 deg.C for use.

(3) Vemurafenib: manufactured by LC Laboratories, Inc.; weighing a certain amount of vemurafenib, dissolving into 10mM vemurafenib solution (mother solution) by DMSO, subpackaging, and placing in a refrigerator at-20 ℃ for later use.

(4) A375 cell line [ human melanoma cells carrying BRAF (V600E) mutation ]: purchased from ATCC cell bank, usa, and stored in liquid nitrogen.

(5) Cells resistant to virofinib acquisition a 375R: a375 cells were cultured in DMEM containing 10% FBS and increasing concentrations (0.1-2. mu.M) of Verofinib for 3 months to give Verofinib resistant melanoma cells A375R, which were subsequently cultured in DMEM containing 2. mu.M Verofinib and 10% FBS to maintain resistance.

(6) DMEM medium powder: manufactured by Gibco corporation of usa.

(7) 0.5% Trypsin-EDTA (10X) (Trypsin) 100 mL/bottle, manufactured by Gibco, USA.

(8) Fetal bovine serum: 500 mL/bottle, manufactured by Gibco, USA.

(9) 96-well flat-bottom culture plates: manufactured by SPL corporation of korea.

(10) CCK8 kit: manufactured by the company Dojindo (Japan).

1.2 Experimental instruments

(1) A carbon dioxide incubator: model nu-4750e, manufactured by Nuaire corporation.

(2) A centrifuge: centrifuge model 5702, manufactured by Eppendorf corporation.

(3) The biological safety cabinet: nu-425 and 400e, manufactured by Nuaire.

(4) An electronic balance: manufactured by Mettler Toledo corporation.

(5) And (3) inverting the microscope: manufactured by Leica corporation.

(6) A water bath device: WNB 14, manufactured by Memmert GmbH + Co.

(7) Microplate reader: manufactured by BIO-RAD corporation.

1.3 Experimental methods and results

Collecting cultured cells with acquired resistance to vemurafenib A375R, adjusting the cell suspension concentration to 3X 10³Adding each well into 96-well culture plate, placing 100 μ L each well at 37 deg.C with 5% CO₂The cell culture box was cultured for 24 hours. The old culture medium was aspirated, 20, 25 μ M of ginsenoside Rg3, 25, 30 μ M of artesunate or a combination of ginsenoside Rg3 and Artesunate (ART) (Rg 3/ART: 20/25, 20/30, 25/30 μ M) was added, and a control group (0.1% DMSO) and a blank group (no cells) were set, each group was set with 4 replicate wells, and the culture was continued for 24 hours. After the end of the administration, 10. mu.L of CCK8 reagent was added to each well, and the OD of each well was measured at 450nm at 1h intervals over 4 h. The OD value is the optical density absorbed by the detected object, and is used for directly reading data on an instrument. The cell survival rate and the effect of drug combination were calculated by the following formulas.

The cell survival rate was (drug-treated OD value-blank OD value)/(control OD value-blank OD value) × 100%.

The ratio of the cell survival rate of the ginsenoside Rg3 and the artesunate composition group to the cell survival rate of the control group/(the ratio of the cell survival rate of the ginsenoside Rg3 group to the cell survival rate of the control group) × (the ratio of the cell survival rate of the artesunate group to the cell survival rate of the control group).

CDI is more than 1, which indicates that the two drugs have antagonistic action;

CDI ═ 1, indicating that the two drugs have additive effects;

CDI <1, which means the two drugs have synergistic effect, wherein CDI <0.7, which means the two drugs have significant synergistic effect.

The results are shown in table 1 and fig. 1.

TABLE 1 Effect of ginsenoside Rg3 and Artesunate on proliferation of melanoma cells resistant to BRAF (V600E) inhibitor alone or in combination

Note: denotes p <0.05 compared to the corresponding control group, and p <0.01 compared to the corresponding control group.

Delta indicates that the drug combination has synergistic effect, and CDI is less than 1.

Delta indicates that the drug combination has obvious synergistic effect, and CDI is less than 0.7.

As can be seen from table 1 and fig. 1, the ginsenoside Rg3 and artesunate can inhibit proliferation of melanoma cells resistant to vemurafenib acquired drug resistance a 375R; the combination of the ginsenoside Rg3 and artesunate has synergistic effect.

Example 2 Effect of ginsenoside Rg3 and Artesunate on Normal cell survival

2.1 Experimental materials

(1) Ginsenoside 20-R-Rg 3: purchased from Nanjing Biotech Ltd; weighing a certain amount of ginsenoside Rg3, dissolving in DMSO to obtain 20mM Rg3 solution (mother liquor), packaging, and refrigerating at-20 deg.C.

(3) Normal cell lines: human immortalized keratinocytes HaCaT; purchased from ATCC cell bank, usa, and stored in liquid nitrogen.

(4) DMEM medium powder: manufactured by Gibco corporation of usa.

(5) 0.5% Trypsin-EDTA (10X) (Trypsin) 100 mL/bottle, manufactured by Gibco, USA.

(6) Fetal bovine serum: 500 mL/bottle, manufactured by Gibco, USA.

(7) 96-well flat-bottom culture plates: manufactured by SPL corporation of korea.

(8) CCK8 kit: manufactured by the company Dojindo (Japan).

2.2 Experimental instruments

(2) A centrifuge: centrifuge model 5702, manufactured by Eppendorf corporation.

(3) The biological safety cabinet: nu-425 and 400e, manufactured by Nuaire.

(4) An electronic balance: manufactured by Mettler Toledo corporation.

(5) And (3) inverting the microscope: manufactured by Leica corporation.

(6) A water bath device: WNB 14, manufactured by Memmert GmbH + Co.

(7) Microplate reader: manufactured by BIO-RAD corporation.

2.3 Experimental methods and results

HaCaT cells cultured in DMEM containing 10% FBS were collected and the cell suspension concentration was adjusted to 4X 10³Adding each well into 96-well culture plate, placing 100 μ L each well at 37 deg.C with 5% CO₂The cell culture box was cultured for 24 hours. Removing old culture medium, 15 μ M ginsenoside Rg3, 10, 15, 20, 50 μ M artesunate or composition of ginsenoside Rg3 and artesunate at different ratios, setting control group (0.1% DMSO) and blank group (no cell), setting 4 multiple wells for each group, and culturing for 48 hr. After the administration, 10. mu.L of CCK8 reagent was added to each well at 4h intervals of 1hThe OD of each well was measured at 50 nm. The OD value is the optical density absorbed by the detected object, and is used for directly reading data on an instrument. The cell viability and the effect of the drug combination were calculated as in example 1.

The results are shown in table 2 and fig. 2.

TABLE 2 Effect of ginsenoside Rg3 and Artesunate on Normal cell survival alone or in combination

Note: ns indicates no significant difference compared to the control group.

As can be seen from Table 2 and FIG. 2, the ginsenoside Rg3 and artesunate alone or in combination have no significant cytotoxic effect on human immortalized keratinocyte HaCaT.

Example 3 Effect of ginsenoside Rg3 in combination with Artesunate on melanoma mice with a Lotus tolerant BRAF (V600E) inhibitor

3.1 Experimental materials

Experimental animals: male BALB/c-nu/nu nude mice (about 8 weeks in size) were provided by the animal testing center, university of Chinese, hong Kong. The feed is raised in a constant-temperature constant-pressure sterile environment in the laboratory animal center of hong Kong university of Dipper.

Cells resistant to virofinib acquisition a 375R: a375 cells were purchased from ATCC cell banks in the united states and vemurafenib resistant melanoma cells a375R were established in example 1, followed by culturing the cells in DMEM containing 2 μ M vemurafenib and 10% FBS to maintain its resistance.

The ginsenoside Rg3 gastric lavage liquid: weighing 7.2mg ginsenoside Rg3, dissolving in 0.3mL DMSO, diluting with sodium carboxymethylcellulose (CMC-Na) to 6mL to obtain 1.2mg/mL ginsenoside Rg3 solution, filtering, and packaging.

Artesunate gastric lavage fluid: weighing 18mg of artesunate, dissolving in 0.3mL of DMSO, diluting to 6mL with 5% CMC-Na solution to obtain 3mg/mL artesunate solution, filtering, and packaging for storage.

Trametinib gastric juice: weighing 10mg trametinib, dissolving in 0.3mL DMSO, diluting with 5% CMC-Na solution to 30mL to obtain 0.33mg/mL trametinib solution, filtering, and subpackaging for storage.

3.2, experimental methods and results:

the cultured cells A375R resistant to the acquired Verofinib were collected, resuspended in Phosphate Buffered Saline (PBS), and inoculated subcutaneously into the back of BALB/c-nu/nu nude mice, each mouse injected with 3X 10⁶Individual cells, scored as day 0. On day 1, mice were randomly divided by body weight into 2 groups, 4 control groups, 4 positive control groups, and 3 drug combination groups, and administered after 8h fasting. The positive control group is administered with trametinib of 0.33 mg/kg/day per day, the drug combination group is administered with ginsenoside Rg3 of 6 mg/kg/day and artesunate of 15 mg/kg/day per day, and the control group is administered with equal volume of solvent (5% CMC-Na solution containing 5% DMSO) for 13 days. The body weight and the tumor volume of the mice are measured at intervals, and a body weight change curve and a tumor volume change curve of the mice are drawn. Mice were sacrificed at the end of the experiment on day 14, solid tumors were detached, tumor weight was weighed, and tumor inhibition rate (%) was calculated. The results are shown in table 3 and fig. 3.

TABLE 3 Effect of ginsenoside Rg3 in combination with Artesunate on Targeted drug resistant melanoma mice with Lotus BRAF (V600E)

Note: denotes p <0.05 compared to control group.

The experimental results showed that the tumor volume growth rate was lower in the positive control group and the drug combination group than in the control group with increasing administration time (fig. 3A). The tumor weight of the tumor-bearing nude mice treated by the combination group of the ginsenoside Rg3 and the artesunate is obviously lower than that of the control group (p <0.05), the inhibition rate of the tumor growth of A375R is 62.24% (table 3, figure 3B), and the weight of the mice is not obviously influenced. The combination of the ginsenoside Rg3 and the artesunate can safely and effectively inhibit the growth of the melanoma which is resistant to the BRAF (V600E) inhibitor in mice.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.

Claims

1. Use of artesunate or its derivatives in the preparation of a medicament for preventing and treating tumors resistant to BRAF (V600E) inhibitors.

2. The application according to claim 1, wherein the derivative of artesunate is a trimeric derivative of artesunate.

3. Use of a composition composed of ginsenoside Rg3 or its derivatives, and artesunate or its derivatives in the preparation of a medicament for treating tumors resistant to BRAF (V600E) inhibitors.

4. The application according to claim 3, wherein the derivative of artesunate is a trimeric derivative of artesunate.

5. The application according to claim 3, wherein the ginsenoside Rg3 is 20-S-Rg3 and/or 20-R-Rg3.

The application according to claim 3, wherein the derivative of ginsenoside Rg3 is 20(R)-25-OH-ginsenoside Rg3 or dihydroginsenoside Rg3.

The application according to claim 3, characterized in that, in terms of mass percentage, the composition comprises: 0.1%-99.9% of ginsenoside Rg3 and/or its derivatives, and 99.9%-0.1% of green Artesunate and/or its derivatives.

8. The application according to any one of claims 1 to 7, wherein the medicine further comprises a pharmaceutically acceptable adjuvant; and/or

The dosage form of the medicine is tablet, capsule, granule, suspension, oral liquid, injection or ointment.

9. The use according to any one of claims 1 to 7, wherein the drug further comprises an anticancer drug.

The application according to any one of claims 1 to 7, wherein the tumor resistant to a BRAF (V600E) inhibitor is an inhibitor targeting BRAF (V600E) or an inhibitor targeting BRAF downstream MEK BRAF(V600E) mutation-positive melanoma, colorectal cancer, prostate cancer, thyroid cancer, non-small cell lung cancer, serous ovarian cancer, or hairy cell leukemia with intrinsic or acquired resistance to the inhibitor.